Uses for prevention or treatment of brain diseases using microrna

ABSTRACT

The present disclosure relates to a pharmaceutical composition for preventing or treating a brain disease, more particularly to a pharmaceutical composition for preventing or treating a brain disease, which contains a miR-485-3p inhibitor, and a method for screening an agent for preventing or treating a brain disease, which includes a step of measuring the expression level of miR-485-3p. Because the composition for treating a brain disease, which contains a miR-485-3p inhibitor, can restore the ELAVL2 protein unlike the exiting therapeutic agents for Alzheimer&#39;s disease, which are limited only to alleviating symptoms by inducing decreased expression of amyloid beta 42, the present disclosure can fundamentally treat various diseases caused by decreased expression of ELAVL2, such as Alzheimer&#39;s disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, etc. Therefore, the present disclosure is useful for treating brain diseases including Alzheimer&#39;s disease fundamentally.

The content of the electronically submitted sequence listing in ASCIItext file (Name: 4366_0120000_SeqListing_ST25.txt; Size: 1,985 bytes;and Date of Creation: Jun. 13, 2019) filed with the application isherein incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to a use of miR-485-3p for preventing ortreating a brain disease, more particularly to a pharmaceuticalcomposition for preventing or treating a brain disease, which contains amiR-485-3p inhibitor, and a method for screening an agent for preventingor treating a brain disease, which includes a step of measuring theexpression level of miR-485-3p.

BACKGROUND OF THE DISCLOSURE

Alzheimer's disease is the most common form of dementia. 75% of patientswith dementia have Alzheimer's disease. In most cases, Alzheimer'sdisease begins in people over 65 years of age, although it can occurearlier in rare cases. In the United States, about 3% of the populationaged 65-74 years, about 19% of the population aged 75-84 years, and 50%of the population aged over 85 years suffer from this disease. In Korea,according to a recently reported study on a rural region, about 21% ofthe population aged over 60 years in the rural region showed dementia,and 63% of them had Alzheimer's dementia. In 2006, 266,000 people aroundthe world had the disease. It is expected that the disease will occur inone out of every 85 people in 2050.

The treatment of Alzheimer's disease has recently focused on the factthat Alzheimer's disease may be caused by impaired cholinergic signalingand transmission in the cerebral cortex and hippocampus (Bartus et al.,Science. 217(4558): 408-14(1982) and Coyle et al., Science. 219(4589):1184-90(1983)).

Because these regions of the brain are associated with memory andintelligence, functional deficit in these regions may cause loss ofmemory and intelligence. Although the process of impairment in neuronalsignaling is still controversial, senile plaques and neurofibrillarytangles (NFT) are considered as main pathological causes.

In particular, development of senile plaques due to the accumulation ofamyloid beta (Aβ) is a notable feature of this disease, and Alzheimer'sdisease can be confirmed by post-mortem examination (Khachaturian, Arch.Neurol. 42(11): 1097-105(1985)).

As a way of treating Alzheimer's disease, a method of increasing theamount of acetylcholine to inhibit the impairment of cholinergicsignaling or causing acetylcholine to act more effectively ontransmission of neuronal cells has been proposed. Thus, patients withAlzheimer's disease use a variety of compounds for increasing theactivity of acetylcholine.

Currently, the most effective way is to rapidly decompose acetylcholinein synapses, thus inhibiting the activity of acetylcholinesterase thatprevents neuronal signaling. These inhibitors (e.g., tacrine, donepezil,galantamine and rivastigmine) are approved by the United States Food andDrug Administration (FDA) and are currently available on the market asAlzheimer's disease medications. Despite their effectiveness inpreventing further destructive progress of this disease, they are notused to cure the disease.

Some compounds are aimed at improving the general state of neurons andmaintaining aged cells in good function. For example, some drugs such asNGF or estrogen act as neuroprotecting agents to delayneurodegeneration, and other drugs such as antioxidants decrease celldamage caused by oxidation of cells resulting from normal aging.

Alzheimer's disease becomes serious as the amyloid beta peptide isaccumulated in the neuritic space. It is thought that the progress ofAlzheimer's disease can be delayed by reducing the accumulation ofamyloid beta. In addition, amyloid precursor protein (APP) is consideredto play a role in combination with proteinases in cells, such as α-, β-and γ-secretases. However, because the process of amyloid beta formationhas not been fully elucidated scientifically, it is not yet possible tocontrol the formation of amyloid beta.

It is not certain how the accumulation of amyloid beta acts on neuronalsignaling. Abnormally cleaved APP induces amyloid beta generation, andplaque formation is induced by the accumulation of amyloid beta in theneuritic space. Thus, various factors involved in this cleavage reaction(e.g., inflammation reaction, etc.) increase the phosphorylation of tauprotein, and also increase the accumulation of paired helical filaments(PHF) in combination with NFT, resulting in damage to the nerve. Allthese factors induce dysfunction of the nerve and, ultimately,accelerates the progress of Alzheimer's disease to dementia.

ELAVL2, or ELAVL-like neuron-specific RNA binding protein 2, is a typeof nELAVL2. nELAVL2 is an RNA-binding protein expressed specifically inthe brain and is known to be associated with neurodegenerative diseases.As a result of conducting high-throughput RNA sequencing using braintissue after post-mortem of patients with Alzheimer's disease, it wasfound out that ELAVL2 was expressed with low levels.

In this regard, U.S. Pat. No. 5,532,219 discloses a composition fortreating Alzheimer's disease containing 4,4′-diaminodiphenylsulfone,etc., U.S. Pat. No. 5,506,097 discloses a composition for treatingAlzheimer's disease containing para-amidinophenylmethanesulfonylfluoride or ebelactone A, and U.S. Pat. No. 6,136,861 discloses acomposition for treating Alzheimer's disease containingbicyclo[2.2.1]heptane.

Recently, the development of therapeutic agents using a microRNAinhibitor is being attempted. WO 2013/045652 (Apr. 4, 2013) discloses atreatment of epilepsy using a miR-134 inhibitor, and WO 2015/025995(Feb. 26, 2015) discloses treatment of epilepsy using a miR-203inhibitor. In addition, European Patent Registration No. 2436784 (Sep.11, 2013) discloses diagnosis and treatment of colon cancer usingmiR-203.

Although the development of therapeutic methods to reduce the effect ofAlzheimer's disease is carried out actively, temporary improvement ofsymptoms is the current strategy. In conclusion, the current treatmentof Alzheimer's disease is just focused on improvement of symptomsinstead of slowing or reversing the progress of the disease. Despite thebiological knowledge about the disease, clinical application is stillnot successful.

Thus, the inventors of the present disclosure have made efforts todevelop an agent for preventing or treating brain diseases includingAlzheimer's disease. As a result, they have confirmed that theinhibition of miR-485-3p expression or the inhibition of interactionbetween miR-485-3p and ELAVL2 leads to inhibition of Aβ42 production,inhibition of APP expression or inhibition of tau proteinphosphorylation, thereby being useful in treating brain diseases, andhave completed the present disclosure.

The information described in the Background section is only to enhancethe understanding of the background of the present disclosure, and theinformation forming the prior art already known to those having ordinaryskill in the art to which the present disclosure belongs may not beincluded.

SUMMARY OF THE DISCLOSURE

The present disclosure is directed to providing a pharmaceuticalcomposition for preventing or treating a brain disease using a microRNA.The present disclosure is also directed to providing a method forscreening an agent for preventing or treating a brain disease bymeasuring the expression level of a microRNA.

In order to achieve the above-described objects, the present disclosureprovides a pharmaceutical composition for preventing or treating a braindisease, which contains a miR-485-3p inhibitor.

The present disclosure also provides a method for preventing or treatinga brain disease, which includes a step of administering apharmaceutically effective amount of a miR-485-3p inhibitor.

The present disclosure also provides a use of a miR-485-3p inhibitor forpreventing or treating a brain disease.

The present disclosure also provides a use of a miR-485-3p inhibitor forpreparing a medication for preventing or treating a brain disease.

The present disclosure also provides a method for screening an agent forpreventing or treating a brain disease, which includes: (A) a step oftreating a cell expressing miR-485-3p with a candidate substance andmeasuring the expression level of miR-485-3p; and (B) a step ofscreening the candidate substance as an agent for preventing or treatinga brain disease if the expression level of miR-485-3p measured in thestep (A) is decreased as compared to a control group not treated withthe candidate substance.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 summarizes a procedure of cDNA synthesis and detection.

FIGS. 2A-2B show a miRNA expression pattern analysis result (volcanoplot) for a patient group as compared to a normal group (FIG. 2A), and amiRNA expression pattern analysis result (scatter plot) for a patientgroup as compared to a normal group (FIG. 2B).

FIG. 3 compares the expression of miR-485-3p in the hippocampus and thecortex.

FIG. 4 shows a list of the 3′-untranslated region (UTR) mRNAs of ELAVL2.

FIGS. 5A-5B shows a comparative quantitative analysis result of Aβ 42 inthe cerebral cortex of 5×FAD (FIG. 5A), and a comparative quantitativeanalysis result of Aβ 42 in the hippocampus (FIG. 5B).

FIG. 6 shows a result of comparing the expression of ELAVL2 in thecerebral cortex and the hippocampus of 5×FAD.

FIGS. 7A and 7B show results of comparing the expression of ELAVL2 (FIG.7A) and Aβ (FIG. 7B) in hippocampal primary cells depending ontransfection with antagomir (AM)-485-3p.

FIG. 8A-8B show an photograph of a mouse brain (FIG. 8A) and an imaginganalysis of drug delivery after intranasal administration ofCy3-AM-485-3p (FIG. 8B).

FIGS. 9A-9B show comparative quantitative analysis results of ELAVL2(FIG. 9A) and Aβ (FIG. 9B) for 5×FAD intranasally treated withAM-485-3p.

FIGS. 10A-10B show a results of comparing the expression of APP (FIGS.10A-10B), tau (FIG. 10B), and p-tau (FIG. 10B) in HeLa cells dependingon AM-485-3p transfection.

FIGS. 11A-11B show results of comparing the cognitive function of 5×FADintranasally treated with AM-485-3p.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by the skilled experts inthe art to which the present disclosure belongs. In general, thenomenclature used herein is known well and commonly used in the art.

In a specific example of the present disclosure, it was confirmed thatthe expression of miR-485-3p is increased in Alzheimer's patients andthat the expression level of ELAVL2 can be recovered and the productionof Aβ 42 can be decreased through an oligonucleotide inhibiting theexpression or activity of miR-485-3p, thereby improving behavioraldisorder and decline in cognitive function, which are the main symptomsof Alzheimer's disease.

Accordingly, in an aspect, the present disclosure relates to apharmaceutical composition for preventing or treating a brain disease,which contains a miR-485-3p inhibitor.

In the present disclosure, the ‘miR’ or ‘microRNA (miRNA)’ refers to anon-coding RNA consisting of 21-23 nucleotides, which is known to beinvolved in post-transcriptional regulation of gene expression bysuppressing the translation of target RNA or promoting degradationthereof.

In the present disclosure, the mature sequence of the miRNA can beobtained from the miRNA database (http://www.mirbase.org). As of Aug.13, 2012, 25,141 mature miRNAs derived from 193 species are listed inthe miRNA database (19th edition, miRBase).

In general, following transcription into a precursor called a pre-miRNA,which has a hairpin structure and is about 70-80 nt (nucleotides) inlength, a mature form of miRNA is produced as the pre-miRNA is cleavedby the RNAse III enzyme Dicer. The miRNA forms a ribonucleoproteincomplex called a miRNP and cleaves a target gene or inhibits itstranslation through complementary binding to the target site. 30% ormore of human miRNAs exist in the form of a cluster.

In the present disclosure, the miR-485-3p may be expressed in the brain,particularly in the hippocampus and the cortex, although not beinglimited thereto. By binding to the 3′-untranslated region of ELAVL2 mRNAwhich encodes ELAVL2 (ELAV-like RNA binding protein 2), it inhibits itsexpression, thereby lowering the concentration of the ELAVL2 protein inthe brain.

In the present disclosure, the sequence of miR-485-3p may be derivedfrom a mammal, for example, a human, mouse or rat. In an exemplaryembodiment of the present disclosure, the sequence of miR-485-3p isderived from a human, and includes not only a mature sequence[5′-GUCAUACACGGCUCUCCUCUCU-3′ (SEQ ID NO 1)] but also a precursorsequence [⁵′-ACUUGGAGAGAGGCUGGCCGUGAUGAAUUCGAUUCAUCAAAGCGAGUCAUACACGGCUCUCCUCUCUUUUAGU-3′ (SEQ ID NO 2)].

In the present disclosure, the miR-485-3p inhibitor may inhibit theexpression of miR-485-3p. Alternatively, it may inhibit the interactionbetween miR-485-3p and the 3′-UTR of ELAVL2 (ELAV-like neuron-specificRNA binding protein 2).

In the present disclosure, the miR-485-3p inhibitor may inhibit orinterfere with the action or function of miR-485-3p in cells. Theinhibition of miR-485-3p includes direct inhibition of binding ofmiR-485-3p to its target, e.g., an mRNA molecule encoding the ELAVL2protein. Also, direct inhibition of the function of miR-485-3p using asmall molecule inhibitor, an antibody or an antibody fragment, orindirect regulation using an inhibitor or a small interfering RNAmolecule is included.

In the present disclosure, the miR-485-3p inhibitor may be a nucleicacid molecule binding to all or a part of the base sequence of SEQ ID NO1 or SEQ ID NO 2.

In the present disclosure, the nucleic acid molecule binding to a partof the base sequence of SEQ ID NO 1 or SEQ ID NO 2 may be 7-50 nt(nucleotides), specifically 10-40 nt, more specifically 15-30 nt,further more specifically 15-25 nt, particularly 16-19 nt, in length,although not being limited thereto.

In the present disclosure, the nucleic acid molecule may bind to the 1stor 2nd through the 7th or 8th base sequence of SEQ ID NO 1.

In the present disclosure, the nucleic acid molecule may be selectedfrom a group consisting of DNA, RNA, an antagomir (antisenseoligonucleotide of miRNA), siRNA, shRNA and an oligonucleotide.

In an exemplary embodiment of the present disclosure, the activity ofthe precursor sequence (SEQ ID NO 2) and the mature sequence (SEQ IDNO 1) is inhibited directly or indirectly for the interference with orinhibition of the activity of miR-485-3p. Also, the inhibition of theactivity of miR-485-3p includes lowering its cellular level byinhibiting the transcription of miR-485-3p and/or the binding ofmiR-485-3p to its target mRNA.

In the present disclosure, the miR-485-3p inhibitor includes anysubstance capable of inhibiting the expression and/or activity ofmiR-485-3p. The substance includes a low-molecular-weight compound, anantagomir, an antisense molecule, a small hairpin RNA (shRNA) molecule,a small interfering RNA (siRNA) molecule, a seed target LNA (lockednucleic acid) oligonucleotide, a decoy oligonucleotide, an aptamer, aribozyme, or an antibody that recognizes a DNA:RNA hybrid, although notbeing limited thereto.

In the present disclosure, the miR-485-3p inhibitor may be an antisenseoligonucleotide which can inhibit the activity of miR-485-3p bycomplementarily binding to all or a part of the precursor and/or maturesequence, particularly the seed sequence. The ‘seed sequence’ is asequence which is very important in recognition of the target moleculeof miRNA and is conserved in a variety of species (Krenz, M. et al., J.Am. Coll. Cardiol. 44: 2390-2397 (2004); H. Kiriazis, et al., Annu. Rev.Physiol. 62: 321 (2000)). Because miRNA binds to its target via thesequence seed, the translation, etc. of the target mRNA may be inhibitedeffectively by inhibiting the interaction between the seed sequence andthe target.

In In the present disclosure, the nucleic acid molecule may be anantisense oligonucleotide containing a sequence all or a part of whichis complementary to the base sequence of SEQ ID NO 1. The antisenseoligonucleotide may be represented by a base sequence selected from agroup consisting of SEQ ID NO 3 to SEQ ID NO 7.

In the present disclosure, the antisense oligonucleotide may include asequence all or a part of which is complementary to the 1st or 2ndthrough the 7th or 8th base sequence of the base sequence of SEQ ID NO1, although not being limited thereto. The antisense oligonucleotide maybe represented by a base sequence selected from a group consisting of5′-GUGUAUGAC-3′ (SEQ ID NO 3), 5′-UGUAUGAC-3′ (SEQ ID NO 4),5′-GUGUAUGA-3′ (SEQ ID NO 5), 5′-UGUAUGA-3′ (SEQ ID NO 6) or5′-AGAGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO 7).

The antisense oligonucleotide includes a nucleic acid-based moleculehaving a sequence complementary to all or a part of a target miRNA,particularly the seed sequence of the miRNA, and thus capable of forminga duplex with the miRNA. Thus, the antisense oligonucleotide may bereferred to as a complementary nucleic acid-based inhibitor.

In addition, the antisense oligonucleotide includes a variety ofmolecules, for example, a ribonucleic acid (RNA), a deoxyribonucleicacid (DNA), an antagomir, a 2′-O-modified oligonucleotide, aphosphorothioate-backbone deoxyribonucleotide, aphosphorothioate-backbone ribonucleotide, a PNA (peptide nucleic acid)oligonucleotide or an LNA (locked nucleic acid) oligonucleotide.Specifically, it may be a ribonucleic acid.

The ribonucleic acid includes a double-stranded small hairpin RNA(shRNA) molecule, a small interfering RNA (siRNA) molecule and aribozyme.

The LNA has a locked conformation due to further modification betweenthe 2 ‘ and 4’ carbon of the ribose moiety of the oligonucleotide and,thus, ensures thermal stability.

The PNA (peptide nucleic acid) contains a peptide-based backbone insteadof a sugar-phosphate backbone.

The 2′-O-modified oligonucleotide is specifically a 2′-O-alkyloligonucleotide, more specifically a 2′-O—C₁₋₃ alkyl oligonucleotide,and most specifically a 2′-O-methyl oligonucleotide.

The antisense oligonucleotide includes an antisense oligonucleotide in anarrow sense, an antagomir and an inhibitory RNA molecule.

The antagomir is a chemically modified single-stranded oligonucleotideand is used to silence an endogenous microRNA. The antagomir contains asequence that is not complementary at the Argonaute 2 (Ago2) cleavagesite, or inhibits cleavage of Ago2 such that the base is modified with,for example, a 2-′methoxy group, a 3′-cholesterol group or aphosphorothioate. There is a complementary sequence to the targetsequence.

In the present disclosure, the antagomir has a sequence which is atleast partially or completely complementary to miR-485-3p. The antagomirmay include one or more modification (e.g., 2′-O-methyl-sugarmodification or 3′-cholesterol modification). Alternatively, theantagomir may contain one or more phosphorothioate linkage and have aphosphorothioate backbone at least in part.

In the present disclosure, the appropriate length of the antagomir forinhibiting the expression of miR-485-3p is 7-50 nt (nucleotides),specifically 10-40 nt, more specifically 15-30 nt, more specifically15-25 nt, more specifically 16-19 nt, although not being limitedthereto.

The term ‘complementary’ as used the present disclosure means that theantisense oligonucleotide is sufficiently complementary to themiR-485-3p target under predetermined hybridization conditions orannealing conditions, specifically under physiological conditions, suchthat it can selectively hybridize to the target, and encompasses bothpartially or substantially complementary and completely (perfectly)complementary. Specifically, it means being completely complementary.Substantially complementary means that, although not completelycomplementary, it has complementarity sufficient to bind to the targetsequence and exert an effect according to the present disclosure, i.e.,interference with the activity of miR-485-3p.

The ‘nucleic acid’ includes an oligonucleotide, a DNA, an RNA, apolynucleotide, and analogs and derivatives thereof. For example, a PNAor a mixture thereof is included. In addition, the nucleic acid may besingle- or double-stranded and can encode molecules including an mRNA, amicroRNA, a siRNA, a polypeptides, etc.

In the present disclosure, the antisense oligonucleotide may include oneor more modification selected from: 1) modification to a LNA (lockednucleic acid) or PNA (peptide nucleic acid) form; 2) substitution of the—OH group at the 2′ carbon of a nucleotide with —CH₃ (methyl); and 3)modification of a nucleotide bond to phosphorothioate.

One or more nucleotide constituting the antisense oligonucleotide may bea LNA or a PNA. The sugar of at least one nucleotide constituting thesame may be 2′-O-methylated or methoxylated, or one or morephosphothioate may be contained in the backbone, although not beinglimited thereto.

In the present disclosure, the miR-485-3p inhibitor may have one or moreof the following features: 1) recovery of the expression level ofELAVL2; 2) inhibition of the production of amyloid beta 42 (Aβ42); 3)inhibition of the expression of amyloid precursor protein (APP); and 4)inhibition of the phosphorylation of tau protein.

In an example of the present disclosure, it was confirmed that themiR-485-3p inhibitor has the features of recovery of the expressionlevel of ELAVL2, inhibition of the production of Aβ42, inhibition of theexpression of APP and inhibition of the phosphorylation of tau proteinusing 5×FAD mouse, which is an animal model of Alzheimer disease whichexhibits severe accumulation of intraneuronal Aβ42 from about 6 weeksdue to overexpression of mutant forms of APP and PSEN1.

It is known that the decreased expression level of ELAVL2 is associatedwith the onset of Alzheimer's disease, autism spectrum disorder, mentalretardation and amyotrophic lateral sclerosis. Especially, it is knownthat the level of the ELAVL2 protein is decreased by substances inducingexcitotoxicity such as kainic acid, NMDA, quisulate, AMPA, glutamate,etc., resulting in neuronal cell death and disturbance of brainfunction, causing a number of brain diseases such as seizure, stroke,Parkinson's disease, spinal cord injury, etc. (Kaminska, B. et al., ActaBiochim Pol. 44: 781-789). Therefore, the recovery of the ELAVL2 proteinthrough the inhibition of the activity of miR-485-3p can be used in thetreatment of various brain diseases such as Alzheimer's disease, autismspectrum disorder, mental retardation, amyotrophic lateral sclerosis,seizure, stroke, Parkinson's disease, spinal cord injury, etc.

In the present disclosure, the brain disease may be selected from agroup consisting of Alzheimer's disease, autism spectrum disorder,mental retardation, amyotrophic lateral sclerosis, seizure, stroke,Parkinson's disease and spinal cord injury, although not being limitedthereto.

In the present disclosure, the pharmaceutical composition may furthercontain, in addition to the miR-485-3p inhibitor, one or more activeingredient exhibiting the same, similar or synergistic function for thetreatment of related diseases or a compound which maintains/increasesthe solubility and/or absorbency of the miR-485-3p inhibitor or theactive ingredient. And, optionally, it may further contain animmunomodulator and/or a chemotherapeutic agent.

The pharmaceutical composition may further contain one or morepharmaceutically acceptable diluent, carrier and/or adjuvant in additionto the above-mentioned active ingredient. As the pharmaceuticallyacceptable carrier, saline, sterile water, Ringer's solution, bufferedsaline, dextrose solution, maltodextrin solution, glycerol, ethanol,liposome, and a mixture of one or more of these components may be used.If necessary, other common additives such as an antioxidant, a buffer, abacteriostatic agent, etc. may be added.

In addition, it can be formulated into an injectable formulation such asan aqueous solution, a suspension, an emulsion, etc., a pill, a capsule,a granule or a tablet by additionally adding a diluent, a dispersant, asurfactant, a binder and a lubricant, and it can be used by binding atarget organ-specific antibody or other ligand with the carrier.

Furthermore, it can be suitably formulated depending on the particulardisease or ingredient by using appropriate methods in the art or usingthe methods disclosed in the Remington's literature (Remington'sPharmaceutical Science (newest edition), Mack Publishing Company, EastonPa.). For example, it can be formulated into one of a suspension, aliposomal formulation, an emulsion, a tablet, a capsule, a gel, a syrupor a suppository.

The pharmaceutical composition may be prepared into a suspension usingan aqueous, nonaqueous or mixed medium. An aqueous suspension mayfurther contain a material increasing the viscosity of the suspension,such as sodium carboxymethyl cellulose, sorbitol and/or dextran.

In the present disclosure, the pharmaceutical composition may beformulated into a formulation for intranasal administration, intravenousadministration, subcutaneous injection, intrathecal injection,inhalation administration or oral administration.

The administration method of the pharmaceutical composition according tothe present disclosure is not particularly limited and any knownadministration method of inhibitors may be applied. Depending onpurposes, parenteral administration (e.g., intranasal, intravenous,subcutaneous, intraperitoneal or topical administration) or oraladministration may be employed. Specifically, administration byintranasal injection may be selected to achieve a quick therapeuticeffect.

The pharmaceutical composition may be delivered via various routes,e.g., via infusion, bolus injection, transdermal or transmucosaladministration (via buccal, anal or intestinal mucosa), or systemic ortopical administration.

In the present disclosure, the pharmaceutical composition may bedelivered to the brain. Specifically, the pharmaceutical composition maybe introduced to the central or peripheral nerves via an appropriateroute. The appropriate route includes intraventricular or intrathecaladministration. The administration may be achieved using a catheterconnected to a reservoir. Also, the pharmaceutical composition may beformulated as an aerosol and may be administered to the lungs using aninhaler or a nebulizer. However, the appropriate route is not limited aslong as the effect of the present disclosure is achieved, and includesintravenous administration, subcutaneous injection, intrathecalinjection, inhalation administration or oral administration.

In the present disclosure, the pharmaceutical composition can beprepared into a variety of unit dosage forms. Such forms include a nasaldrop, a nasal spray, a nasal gel, a nasal ointment and a nasal powder,although not being limited thereto.

In an exemplary example of the present disclosure, the composition couldbe administered intranasally. The effect of the pharmaceuticalcomposition can be enhanced when it is administered intranasally becauseit is delivered to the brain through the olfactory pathway. The nasalcavity refers to a space in the nose, which is divided into left andright fossae by the nasal septum, and the intranasal administrationrefers to delivery of the composition of the present disclosure to anytissue of the nasal epithelium. For the intranasal administration, anintranasally acceptable carrier may be contained. The carrier refers toone or more solid or liquid filler, diluent or encapsulating materialwhich is suitable for administration to any portion of the nasalepithelium of a mammal, specifically human. Typically, the carrier maybe a liquid, a solution, a suspension, a gel, an ointment, a lotion, ora combination thereof. Specifically, the carrier may be apharmaceutically acceptable aqueous carrier.

In addition, the carrier may contain a delivery-enhancing agent. Anintranasal delivery-enhancing agent may include anaggregation-inhibiting agent, a dosage-changing agent, a pH controlagent, a degradative enzyme-inhibiting agent, a mucolytic ormucus-clearing agent, a ciliostatic agent, a membranepenetration-enhancing agent, a surfactant, a bile salt, a phospholipidor fatty acid additive, a mixed micelle, a liposome or carrier, analcohol, an enamine, a nitric oxide-donating compound, a long-chainamphiphilic molecule, a small hydrophobic penetration enhancer, a sodiumor salicylic acid derivative, a glycerol ester of acetoacetic acid, acyclodextrin or beta-cyclodextrin derivative, a medium-chain fatty acid,a chelating agent, an amino acid or a salt thereof, a N-acetylamino acidor a salt thereof, a degradative enzyme for a selected membranecomponent, a fatty acid synthesis inhibitor, a cholesterol synthesisinhibitor, a nitric oxide-stimulating material, a modulatory agent ofepithelial junction physiology such as chitosan or a chitosanderivative, a vasodilator, a selective transport-enhancing agent, etc.In order to enhance intranasal mucosal delivery, a stabilizing deliveryvehicle, carrier, support, complex-forming species, etc. which allowseffective combination, association, storage and encapsulation of thecomposition of the present disclosure and stabilizes the activeingredient may be contained.

In the present disclosure, the pharmaceutical composition may beadministered in a pharmaceutically or therapeutically effective amount.The pharmaceutically or therapeutically effective amount means an amountsufficient to treat a disease at a reasonable benefit/risk ratioapplicable to medical treatment, and an effective dose level will dependon factors including the type and severity of the disease, the activityof a drug, sensitivity to the drug, the time of administration, theroute of administration, the rate of excretion, the duration of thetreatment, and drugs used together, and other factors well known in themedical field.

In addition, the pharmaceutical composition may be administered as anindividual therapeutic agent or in combination with other therapeuticagents, sequentially or concurrently with conventional therapeuticagents, and may be administered singly or multiply. It is important thatthe pharmaceutical composition is administered in such an amount thatthe maximum effect can be obtained with a minimum amount without sideeffects considering all of the above-mentioned factors, which can beeasily determined by those skilled in the art.

The dosage may vary depending on the patient's body weight, age, sex,health condition and diet, administration time, administration method,excretion rate, the severity of the disease, etc., and a proper dosagemay also vary depending on the amount of the drug accumulated in thepatient's body and/or the specific efficacy of the polynucleotide used.In general, it can be calculated on the basis of EC₅₀ measured aseffective from an in-vivo animal model and in vitro. For example, it maybe from 0.01 μg to 1 g per 1 kg of body weight, and may be administeredonce to several times per unit period in a daily, weekly, monthly, orannual unit period. Also, it can be administered continuously for a longperiod of time using an infusion pump. The number of repeatedadministrations is determined in consideration of the time during whichthe drug remains in the body, the drug concentration in the body, andthe like. Even after treatment according to the course of diseasetreatment, the pharmaceutical composition can be continuouslyadministered to prevent the recurrence of the disease.

In the present disclosure, the active ingredient of the pharmaceuticalcomposition, e.g., the antisense oligonucleotide, can be used in thecomposition as it is or in the form of a pharmaceutically acceptablesalt. The pharmaceutically acceptable salt refers to a salt that retainsthe desired biological activity of the oligonucleotide according to thepresent disclosure and exhibits minimal undesired toxicological effect.The salt includes, for example, a base addition salt formed with a metalcation such as zinc, calcium, bismuth, barium, magnesium, aluminum,copper, cobalt, nickel, cadmium, sodium, potassium, etc., or a saltformed with a cation derived from ammonia, N,N-dibenzylethylenediamine,D-glucosamine, tetraethylammonium or ethylenediamine, although not beinglimited thereto.

In the present disclosure, the antisense oligonucleotide, which is theactive ingredient of the pharmaceutical composition, may be negativelycharged due to the characteristic of the nucleotide. The cellular uptakeof the antisense oligonucleotide may be reduced due to the lipophilicnature of cell membranes. The hindered uptake due to polarity can beavoided by using the prodrug approach described in Crooke, R. M. (1998)in Crooke, S. T. Antisense research and Application. Springer-Verlag,Berlin, Germany, vol. 131, pp. 103-140.

The term ‘improvement’, ‘treatment’, or ‘alleviation’ as used in thepresent disclosure means any action to change favorably or improve thesymptoms of related diseases by administering the composition. Those ofordinary skill in the art to which the present disclosure belongs willknow the exact criteria of diseases by referring to the data presented,for example, by the Korean Academy of Medical Sciences and will be ableto judge the degree of improvement, progress and treatment.

The term “prevention” used in the present disclosure means any action toinhibit or delay the onset of related diseases. It will be apparent tothose skilled in the art that the related diseases can be prevented ifthe pharmaceutical composition according to the present disclosure isadministered when or before early symptoms appear.

In an example of the present disclosure, it was confirmed that theexpression of miR-485-3p is increased in Alzheimer's patients and thatbehavioral disorder and decline in cognitive function, which are themain symptoms of Alzheimer's disease, can be improved by anoligonucleotide which inhibits the expression or activity of miR-485-3p.

Accordingly, in another aspect, the present disclosure relates to amethod for preventing or treating a brain disease, which includes a stepof administering a pharmaceutically effective amount of a miR-485-3pinhibitor.

In the present disclosure, the method for preventing or treating a braindisease inhibits the activity of miR-485-3p in the cells or tissues,particularly in the brain cells or brain tissues, of a subject.

In another aspect, the present disclosure relates to a use of amiR-485-3p inhibitor for preventing or treating a brain disease.

In another aspect, the present disclosure relates to a use of amiR-485-3p inhibitor for preparing a medication for preventing ortreating a brain disease.

Reference can be made to the above description regarding the miR-485-3pinhibitor, the regulation or inhibition of the activity of miR-485-3p,administration method, diseases that can be treated, etc.

In another aspect, the present disclosure relates to a method forscreening an agent for preventing or treating a brain disease, whichincludes: (A) a step of treating a cell expressing miR-485-3p with acandidate substance and measuring the expression level of miR-485-3p;and (B) a step of screening the candidate substance as an agent forpreventing or treating a brain disease if the expression level ofmiR-485-3p measured in the step (A) is decreased as compared to acontrol group not treated with the candidate substance.

In the present disclosure, the activity of miR-485-3p may be determinedby analyzing the interaction between miR-485-3p and the 3′-UTR of ELAVL2(ELAV-like neuron-specific RNA binding protein 2).

In the present disclosure, the brain disease may be selected from agroup consisting of Alzheimer's disease, autism spectrum disorder,mental retardation, amyotrophic lateral sclerosis, seizure, stroke,Parkinson's disease and spinal cord injury.

In the screening method of the present disclosure, after contacting acell expressing miR-485-3p with candidate substances, the change in theexpression level of miR-485-3p may be compared with that before thecontacting or with a control group cell not in contact with the testsubstances and the substance which shows change, particularly decrease,in the expression level may be selected as an agent for preventing ortreating a brain disease.

The expression level of miR-485-3p may be measured by performing a knownmethod such as northern blot, RT-PCR, a hybridization method using amicroarray, etc.

In the present disclosure, the miR-485-3p is provided in the form of acell expressing the same, and the activity is determined by analyzingthe interaction between miR-485-3p and the 3′-UTR of its target ELAVL2protein. For example, after contacting a cell expressing the miR-485-3paccording to the present disclosure with candidate substances, thechange in the expression level of miR-485-3p may be compared with thatbefore the contacting or with a control group cell not in contact withthe test substances and the substance which shows change, particularlydecrease, in the expression level may be selected as an agent forpreventing or treating a brain disease.

In the present disclosure, the type of the cell and the amount and kindof the candidate substance used in the screening method will varydepending on the particular test method and candidate substance used,and those skilled in the art will be able to select the suitable type,amount and/or condition of the cell. Based on the test result, thesubstance which leads to decreased activity of miR-485-3p in thepresence of the test substance as compared to the control group not incontact with the test substance is selected as a therapeutic agent. Thedecrease means decrease by about 99% or less, decrease by about 95% orless, decrease by about 90% or less, decrease by about 85% or less,decrease by about 80% or less, decrease by about 75% or less, decreaseby about 70% or less, decrease by about 65% or less, decrease by about60% or less, decrease by about 55% or less, decrease by about 50% orless, decrease by about 45% or less, decrease by about 40% or less,decrease by about 30% or less, or decrease by about 20% or less, ascompared to the control group, although not being limited thereto.

The RNA-RNA interaction used in the screening method according to thepresent disclosure may be detected by a method known in the art, forexample, RNA walk (Lusting et al., Nucleic Acids Res. 2010; 38 (1): e5)or yeast two-hybrid system (Piganeau et al, RNA 2006; 12: 177-184, andRNA: A Laboratory Manual (Cold Spring Harbor Laboratory Press 2011)).

The candidate substance means a substance which is expected to inhibitthe activity of miR-485-3p, and includes a low-molecular-weightcompound, a high-molecular-weight compound, a mixture of compounds(e.g., a natural extract or a cell or tissue culture), a biomedicine(e.g., a protein, an antibody, a peptide, DNA, RNA, an antisenseoligonucleotide, RNAi, an aptamer, RNAzyme and DNAzyme), a sugar and alipid, although not being limited thereto. The candidate substance canbe a polypeptide having two or more amino acid residues, for example, 6,10, 12, 20 or fewer, or more than 20, e.g., 50, amino acid residues. Thecandidate substance may be obtained from a library of synthetic ornatural compounds, and a method for obtaining a library of suchcompounds is known in the art. The libraries of synthetic compounds arecommercially available from Maybridge Chemical Co. (UK), Comgenex (USA),Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), andthe libraries of natural compounds are commercially available from PanLaboratories (USA) and MycoSearch (USA). The test substance may beobtained by a variety of combinatorial library methods known in the art,for example, a biological library, a spatially addressable parallelsolid-phase or solution-phase library, a synthetic library requiringdeconvolution, a “one-bead/one-compound” library, and a syntheticlibrary using affinity chromatography selection. Method for thesynthesis of molecular libraries are disclosed in DeWitt et al., Proc.Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad.Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678,1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem.Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed.Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, or thelike.

In the present disclosure, a low-molecular-weight compound exhibiting atherapeutic effect may be used for the screening purpose of a drug whichtreats a brain. For example, a compound with a molecular weight of about1000 Da, e.g., 400 Da, 600 Da or 800 Da, may be used. Depending onpurposes, these compounds can form a part of a compound library, and thenumber of compounds that make up the library can also vary from dozensto millions. The compound library may contain peptides, peptoids, othercyclic or linear oligomeric compounds, template-basedlow-molecular-weight compounds, e.g., benzodiazepines, hydantoins,biaryls, carbocycles and polycyclic compounds (e.g., naphthalene,phenothiazine, acridine, steroid, etc.), carbohydrates, amino acidderivatives, dihydropyridines, benzhydryls and heterocycles (e.g.triazine, indole, thiazolidine, etc.), although not being limitedthereto.

Also, biologics may be used for the screening. The biologics refers touse of a cell or a biomolecule, and the biomolecule refers to a protein,a nucleic acid, a carbohydrate, a lipid or a material produced in vivoor in vitro using a cellular system. The biomolecule may be providedeither alone or in combination with other biomolecules or cells. Forexample, the biomolecule includes polynucleotides, peptides, antibodiesor other proteins or biological organic materials found in the plasma.

Hereinafter, the present disclosure will be described in detail throughexamples. However, the following examples are for illustrative purposesonly and it will be apparent to those of ordinary skill in the art thatthe scope of the present disclosure is not limited by the examples.

Example 1: Analysis of miRNA Expression Pattern in Alzheimer's PatientsUsing Microarray

(1) Patients and Sample Preparation

Table 1 shows the characteristics of the patients used in the study.About 3 mL of blood was collected in blood tubes (Becton Dickinson,Germany) containing sodium citrate (3.2% w/v) from 4 patients diagnosedwith Alzheimer's dementia by physicians. Four healthy adults ofcorresponding ages (±4 years) were included as a control group.

TABLE 1 Sex and age of normal group and patient group Group Sample No.Sex Age Normal group N1 Female 78 Normal group N2 Male 72 Normal groupN3 Female 74 Normal group N4 Male 79 Patient Group S1 Female 72 PatientGroup S2 Female 82 Patient Group S3 Female 84 Patient Group S4 Male 75

The blood was centrifuged for 10 minutes at 3,500 rpm to separate plasmaand then stored at −80° C. until RNA extraction. miRNA was extractedusing the miRNAeasy Serum/Plasma kit (Qiagen, USA) according to themanufacturer's recommendations. The concentration and purity of theextracted RNA were analyzed using Bioanalyzer 2100 (Agilent, USA). Eightgroups including a normal group satisfied the quality criteria and wereused in the study.

(2) Microarray Screening

Table 2 shows a list of genes used in microarray assay. The maturesequence of each miRNA is available from the miRNA database(http://www.mirbase.org). The extracted RNA was screened using a miRNAarray containing 84 different miRNAs known to be associated with humanneurological development and the progress of neurological disease.

TABLE 2 List of genes used in miRNA qPCR array assay No. Mature miRNA1ist 1 hsa-let-7b-5p 2 hsa-let-7c-5p 3 hsa-let-7d-5p 4 hsa-let-7e-5p 5hsa-let-7i-5p 6 hsa-miR-101-3p 7 hsa-miR-105-5p 8 hsa miR 106b 5p 9hsa-miR-107 10 hsa-miR-124-3p 11 hsa-miR-125b-5p 12 hsa-miR-126-5p 13hsa-miR-128-3p 14 hsa-miR-130a-3p 15 hsa miR 132 3p 16 hsa-miR-133b 17hsa-miR-134-5p 18 hsa-miR-135b-5p 19 hsa-miR-138-5p 20 hsa-miR-139-5p 21hsa-miR-140-5p 22 hsa-miR-146a-5p 23 hsa-miR-146b-5p 24 hsa-miR-148b-3p25 hsa-miR-151a-3p 26 hsa-miR-152-3p 27 hsa-miR-15a-5p 28 hsa-miR-15b-5p29 hsa-miR-181a-5p 30 hsa-miR-181d-5p 31 hsa-miR-191-5p 32hsa-miR-193b-3p 33 hsa-miR-195-5p 34 hsa miR 19b 3p 35 hsa miR 203a 3p36 hsa-miR-20a-5p 37 hsa-miR-212-3p 38 hsa miR 22 3p 39 hsa miR 24 3p 40hsa-miR-26b-5p 41 hsa-miR-27a-3p 42 hsa miR 28 5p 43 hsa-miR-298 44hsa-miR-29a-3p 45 hsa-miR-29b-3p 46 hsa-miR-29c-3p 47 hsa-miR-302a-5p 48hsa-miR-302b-5p 49 hsa-miR-30d-5p 50 hsa miR 320a 51 hsa-miR-328-3p 52hsa-miR-337-3p 53 hsa-miR-338-3p 54 hsa-miR-339-5p 55 hsa-miR-342-3p 56hsa-miR-346 57 hsa miR 34a 5p 58 hsa-miR-376b-3p 59 hsa-miR-381-3p 60hsa-miR-409-3p 61 hsa-miR-431-5p 62 hsa-miR-432-5p 63 hsa-miR-433-3p 64hsa-miR-455-5p 65 hsa-miR-484 66 hsa-miR-485-3p 67 hsa-miR-485-5p 68hsa-miR-487a-3p 69 hsa-miR-488-3p 70 hsa-miR-489-3p 71 hsa-miR-499a-5p72 hsa-miR-509-3p 73 hsa-miR-511-5p 74 hsa-miR-512-3p 75 hsa-miR-518b 76hsa miR 539 5p 77 hsa miR 652 3p 78 hsa-miR-7-5p 79 hsa-miR-9-5p 80 hsamiR 9 3p 81 hsa miR 92a 3p 82 hsa-miR-93-5p 83 hsa-miR-95-3p 84 hsa miR98 5p

FIG. 1 summarizes the procedure of cDNA synthesis and detection. Thequantitative PCR assay method can be summarized as follows. A maturemiRNA is generally a 22-nt, non-coding RNA and is responsible forpost-transcriptional regulation. Polyadenylation of mature miRNA wasinduced by poly(A) polymerase, and cDNA was synthesized using oligo-dTprimers. The oligo-dT primer enables the amplification of the maturemiRNA during the real-time PCR process because it has a 3′ degenerateanchor and a universal tag sequence at the 5′ end. The mature miRNA wasquantified during the real-time PCR process using the miScript SYBRGreen PCR kit (Qiagen).

(3) Analysis of miRNA Expression Pattern Through Volcano Plot

FIG. 2A shows a miRNA expression pattern analysis result (volcano plot)for the patient group as compared to the normal group, and FIG. 2B showsa miRNA expression pattern analysis result (scatter plot) for thepatient group as compared to the normal group. The expression pattern of84 miRNAs was analyzed as compared to the normal group.

The x axis represents fold-change and the y axis represents −log 10 ofthe p value. The horizontal black line shows where the p value is 0.05or smaller. As a result of the volcano plot analysis, it was confirmedthat the expression of hsa-miR-105-5p, hsa-miR-98-5p, hsa-miR-15a-5p,hsa-miR-134-5p, hsa-miR-409-3p, hsa-miR-19b-3p, hsa-miR-92a-3p,hsa-miR-28-5p, hsa-miR-30d-5p, hsa-miR-212-3p, hsa-miR-93-5p,hsa-miR-342-3p, hsa-miR-381-3p, hsa-miR-431-5p, hsa-miR-130a-3p,hsa-miR-146b-5p, hsa-miR-29a-3p, hsa-miR-132-3p, hsa-miR-376b-3p,hsa-miR-22-3p, hsa-miR-509-3p, hsa-miR-139-5p, hsa-miR-499a-5p,hsa-miR-203a-3p, hsa-miR-95-3p, hsa-miR-128-3p, hsa-miR-487a-3p,hsa-miR-485-3p, hsa-miR-195-5p, hsa-miR-433-3p, hsa-miR-133b,hsa-miR-191-5p, hsa-miR-489-3p, hsa-miR-432-5p, hsa-miR-29c-3p,hsa-miR-485-5p, hsa-miR-652-3p, hsa-miR-126-5p, hsa-miR-328-3p,hsa-let-7b-5p, hsa-miR-539-5p, hsa-miR-106b-5p, hsa-miR-101-3p,hsa-miR-302a-5p, hsa-miR-484, hsa-miR-518b, hsa-miR-148b-3p,hsa-miR-181d-5p, hsa-miR-7-5p, hsa-miR-512-3p, hsa-miR-151a-3p,hsa-miR-15b-5p, hsa-let-7e-5p, hsa-miR-135b-5p, hsa-miR-181a-5p,hsa-miR-138-5p, hsa-miR-34a-5p, hsa-miR-346, hsa-miR-511-5p,hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-487a-3p, hsa-miR-489-3p,hsa-miR-499a-5p, hsa-miR-509-3p, hsa-miR-511-5p, hsa-miR-512-3p,hsa-miR-518b, hsa-miR-539-5p, hsa-miR-652-3p, hsa-miR-7-5p,hsa-miR-92a-3p, hsa-miR-93-5p hsa-miR-95-3p and hsa-miR-98-5p wasincreased in the patient group. However, the regulation of miRNA was notstatistically significant except for hsa-miR-485-3p. The expression ofhsa-485-3p was significantly increased as compared to the normal group,with a p value of 0.00439. Therefore, hsa-miR-485-3p can be used as amarker for treatment of Alzheimer's disease.

Table 3 shows the base sequence of has-miR-485-3p. Based on the aboveresult, a functional study was conducted to elucidate the physiologicalfunctions of has-miR-485-3p on cells by synthesizing the sequence.

TABLE 3 Base sequence of hsa-miR485-3p Gene Sequence (5′ → 3′) SEQ ID NOhsa-miR485-3p GUCAUACACGGCUCUCCUCUCU 1

Example 2: Analysis of miR-485-3p Expression in Hippocampus and CerebralCortex of 5×FAD Mouse (RT-qPCR)

(1) Research Methods

The 5×FAD transgenic mouse is an animal model of Alzheimer diseaseobtained by overexpressing mutant forms of APP and PSEN1, which exhibitssevere accumulation of intraneuronal Aβ42 from about 6 weeks.

Given the results of Example 1, RT-qPCR was performed to confirm theexpression of miR-485-3p in the dementia animal model. 5×FAD transgenicmice and wild-type (WT) mice were deeply anesthetized and sacrificed bydecapitation. After excising the brain immediately, the hippocampus andcerebral cortex were dissected from the remaining brain structure. TotalmiRNA was isolated from the hippocampus using the PAXgene Tissue miRNAkit (Qiagen, USA) according to the manufacturer's instructions. cDNA wassynthesized using the miScript II RT kit (Qiagen, USA), and qPCR wasperformed using the mmu_miR-485-3p miScript Primer Assay kit and themiScript SYBR Green PCR kit. The miRNA level was quantified bynormalizing to snoRNA202 (control mouse).

(2) Research Results

FIG. 3 compares the expression of miR-485-3p in the hippocampus and thecortex. RT-PCR was conducted to investigate the expression pattern ofmiR-485-3p in the hippocampus and the cerebral cortex of 5×FAD. Theresult showed that the expression of miR-485-3p was increased in thehippocampus of 5×FAD as compared to WT. This, together with the resultsof Example 1, shows that the expression of miR-485-3p is increased inAlzheimer's dementia. Therefore, the neuronal target mRNA or proteinthat may be affected by miR-485-3p was investigated.

Example 3: Prediction of Target Gene of miR-485-3p

In order to analyze the base sequence and target location ofhsa-miR-485-3p, it was confirmed using a target prediction software(miRDB) that the 3′-untranslated region (UTR) of human-derived ELAVL2 isthe target of hsa-miR-485-3p. It was confirmed that the identified seedsequence was conserved also in mmu-miR-485-3p and the 3′-untranslatedregion of mouse-derived ELAVL2.

FIG. 4 shows a list of the 3′-untranslated region (UTR) mRNAs of ELAVL2,and shows the target 3′-untranslated region (UTR) mRNAs of miR485-3p.The 5′ seed sequence of miR-485-3p (ELAVL2) is shown in blue color.Table 4 shows the base sequence and target location of mmu-miR485-3p. Itwas confirmed using a target prediction software (miRDB) that the3′-untranslated region (UTR) of human-derived ELAVL2 is the target ofmmu-miR-485-3p.

TABLE 4 Analysis of base sequence and target location of mrnu-miR485-3pGene Sequence (5′ → 3′)

mmu-miR-485-3p AGUCAUACACGGCUCUCCUCUC SEQ ID NO Target TotalRepresentative gene Gene name 3P-seq tags+5 sites miRNA ELAVL2ELAV like neuron-specific 78 3 mmu-miR-485-3p RNA binding protein 2

Example 4: Confirmation of Expression of Amyloid Beta (Aβ) 42 and ELAVL2in Hippocampus and Cerebral Cortex of 5×FAD Mouse

(1) Research Methods

Given the results of Example 3, the expression of Aβ 42 and ELAVL2 inthe hippocampus and the cerebral cortex of 5×FAD was investigated. Aftersacrificing an anesthetized mouse by decapitation, the brain wasextracted immediately. After preparing a homogenate of the brain(hippocampus and cerebral cortex), western blot was conducted usinganti-ELAVL2 antibody (Abcam, USA). The immunoreactive protein wasvisualized with a chemiluminescence reagent (GE Healthcare, UK) and wasmeasured and quantified using a chemiluminescence analyzer (Fusion SL).Aβ 42 in the hippocampus and the cerebral cortex was quantified by usingthe mouse/rat amyloid beta (1-42) ELISA kit (IBL) according to themanufacturer's instructions.

(2) Research Results

1) Confirmation of Aβ 42 Expression in Hippocampus and Cerebral Cortex

FIGS. 5A-5B show results of quantitatively comparing the expression ofAβ42 in the cerebral cortex (FIG. 5A) and the hippocampus (FIG. 5B) of5×FAD. It was confirmed that Aβ42 was significantly increased ascompared towild-typ (WT) both in the cerebral cortex and in thehippocampus.

2) Confirmation of ELAVL2 Expression in Hippocampus and Cerebral Cortex

FIG. 6 shows the expression of ELAVL2 in the cerebral cortex andhippocampus of 5×FAD. ELAVL2, an ELAV-like RNA-binding protein, is knownas a protein that regulates neural functions such as neuronal excitationor synaptic transmission, which are directly associated with cognitiveand behavioral functions. From FIG. 6, it was confirmed that theexpression of ELAVL2 in the hippocampus and cerebral cortex of 5×FAD wasdecreased as compared to WT. This suggests that the dementia induced in5×FAD is associated with the decline in cognitive and behavioralfunctions caused by decreased ELAVL2.

Example 5: Preparation of Hippocampal Primary Cell Line and In-VitroTransfection with Antagomir (AM)-485-3p

(1) Research Methods

Primary cells derived from the tissues of the hippocampus and thecerebral cortex excised from the embryo of 5×FAD were cultured. Themethods for cell preparation and culture followed the previous research(Seibenhener, M. L & Woonten M. W, Isolation and culture of HippocampalNeurons from Prenatal Mice, Jove, 2012). 50 nM of miR-485-3p duplex (orscrambled miRNA duplex; Bioneer, Daejon, South Korea) and 50 nM ofantagomir (AM) 485-3p were transfected into primary cells in vitro usingLipofectamine 2000. A cell homogenate was obtained 48 hours after thetransfection, which was subjected to western blot using ELAVL2 antibody(Abcam, UK). The immunoreactive protein was visualized with achemiluminescence reagent (GE Healthcare, UK) and was measured andquantified using a chemiluminescence analyzer (Fusion SL). The amyloidbeta 42 protein was measured by using the mouse/rat amyloid beta (1-42)ELISA kit (IBL) according to the manufacturer's instructions.

(2) Research Results

FIGS. 7A-7B show the results of comparing the expression of ELAVL2 andAP depending on transfection of hippocampal primary cells with AM-485-3p(2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)).

It was confirmed that ELAVL2 was expressed in the hippocampal primarycells of 5×FAD, and the expression of ELAVL2 was increased in the cellstransfected with antagomir (AM)-485-3p as compared to the control (FIG.7 A). This means that miR-485-3p inhibits the expression of ElAVL2 inthe cells treated with the antagomir. Because ELAVL2 is an importantfactor affecting cognitive function by being involved in excitation ofneurons, the development of a drug or a composition that increasesELAVL2, such as a miR-485-3p inhibitor, can be a key strategy inpreventing or treating Alzheimer's disease.

Also, it was confirmed that the expression of Aβ 42 was decreased in thecells transfected with AM-485-3p((2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9); FIG. 7B).This means that miR-485-3p affects the production of Aβ 42 and suggeststhat the development of a drug or a composition capable of inhibitingmiR-485-3p can relieve the pathological symptoms of Alzheimer's dementiaby inhibiting the accumulation of Aβ.

Example 6: Imaging Analysis of Drug Delivery after IntranasalAdministration of Cy3-AM-485-3p

(1) Research Methods

The inhibition of miR-485-3p was induced by intranasally administering asequence-specific antagomir. The intranasal administration of theantagomir was carried out according to a method targeting the brainwithout anesthetizing the mouse (Leah R. T., et al. (2013) IntranasalAdministration of CNS Therapeutics to Awake Mice. J Vis Exp. 2013; (74):4440). After immobilizing the accustomed mouse for intranasal inhalation(intranasal grip) and positioning so that the abdomen faces upward, apipette was positioned in front of one nasal cavity. 6 μL was inhaleddropwise twice using the pipette (1 drop=3 μL). After maintaining theposition for 15 seconds, intranasal inhalation into the right nasalcavity was conducted in the same manner. The same procedure was repeated2 minutes later. A total of 24 μL was inhaled (AM485(2′-O-methylated)-5′-gagaggagagccguguaugacu-3′ (SEQ ID NO 9); 5 nmol in24 μL of distilled water treated with 0.1% v/v diethylpyrocarbonate;Bioneer, Korea). A vehicle of the same volume was administered to acontrol mouse. 12 weeks after the nasal administration, the anesthetizedmouse was sacrificed by decapitation and the brain was excisedimmediately. After fixing the sagittally sectioned brain tissue, thetissue was treated with DAPI to stain DNA. The stained sample was imagedusing a confocal laser scanning microscope (LSM510).

(2) Research Results

As a result of intranasally administering the AM-485-3p(2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9))fluorescence-labeled with Cy3, it was confirmed that the target neuronswere stained with DAPI (FIG. 8B).

Example 7: Comparative Quantitative Analysis of ELAVL2 and AO in 5×FADIntranasally Treated with Antagomir (AM)-485-3p

(1) Research Methods

The intranasal administration of AM-485-3p(2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)) wasconducted as described in Example 6 (Lee, S. T. et al. (2012) miR-206regulates brain-derived neurotrophic factor in Alzheimer disease model.Ann Neurol, 72, 269-277). For intranasal administration of theantagomir, an anesthetized mouse was placed in supine position with itshead flat on the surface. AM-485(2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9); 5 nmol in24 μL of distilled water treated with 0.1% v/v diethylpyrocarbonate;Bioneer, Korea) was administered with a pipette while alternating naresevery 2 minutes, with 4 μL per each administration (6 times in total). Avehicle of the same volume was administered to a control mouse. On day 7after the intranasal administration, the anesthetized mouse wassacrificed by decapitation and the brain was excised immediately. Afterpreparing a homogenate of the brain (hippocampus and cerebral cortex),western blot was conducted using ELAVL2 antibody (Abcam, USA). Theimmunoreactive protein was visualized with a chemiluminescence reagent(GE Healthcare, UK) and was measured and quantified using achemiluminescence analyzer (Fusion SL). Aβ 42 was measured by using themouse/rat amyloid beta (1-42) ELISA kit (IBL) according to themanufacturer's instructions.

(2) Research Results

FIGS. 9A-9B show the comparative quantitative analysis of ELAVL2 and Aβin 5×FAD intranasally treated with the AM-485-3p(2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)). Because itwas confirmed that the treatment of a mouse primary cell line withAM-485-3p induces change in ELAVL2 and Aβ (Example 5), the effect ofAM-485-3p in vivo was investigated by intranasally treating 5×FAD withAM-485-3p. The AM-485-3p group showed increased expression of ELAVL2 ascompared to the control group (FIG. 9 A). This suggests that theexpression of ELAVL2 is decreased as the expression of miR-485-3p isincreased, and that the decreased level of ELAVL2 can be increased bytreating with a miR-485-3p inhibitor such as AM-485-3p.

In addition, since it was confirmed in the animal model that thetreatment with AM-485-3p affects the inhibition of Aβ42 production (FIG.9 B), it can be seen that treatment with the related inhibitor or drugcan relieve the pathological symptoms of Alzheimer's dementia.

Example 8: Expression Pattern of APP and Pattern of Tau and p-Tau inHeLa Cells Stably Transfected with Swedish Mutant Form of AβPP (AβPPsw)Depending on Treatment with AM-485-3p

(1) Research Methods

HeLa cells in which AβPPsw was expressed stably were transfected with5-500 or 50 nM of miR-485-3p duplex (or scrambled miRNA duplex; Bioneer,Daej on, South Korea) and 50 nM of antagomir (AM)-485-3p in vitro usingLipofectamine 2000. A cell homogenate was obtained 48 hours after thetransfection, which was subjected to western blot using APP antibody(Cell Signaling, USA), Tau (Thermofisher Scientific) and p-Tau(Thermofisher Scientific). The immunoreactive protein was visualizedwith a chemiluminescence reagent (GE Healthcare, UK) and was measuredand quantified using a chemiluminescence analyzer (Fusion SL).

(2) Research Results

The expression of APP and pattern of Tau and p-Tau in the HeLa cellsdepending on the treatment with AM-485-3p(2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)) wascompared (FIGS. 10A-10B). It was confirmed that the expression of APP isdecreased in the cells transfected with AM-485-3p in aconcentration-dependent manner as compared to the control group. Inaddition, it was confirmed that the HeLa cell treated with 50 nM ofAM-485-3p showed decreased phosphorylation of tau protein, which isknown as another cause of Alzheimer's disease. This suggests that thedevelopment of a drug such as a miR-485-3p inhibitor or a compositionthereof can be a key strategy in preventing or treating Alzheimer'sdisease by inhibiting the precursor of amyloid beta and thephosphorylation of tau protein, which are known as main causes ofAlzheimer's disease, at the same time.

Example 9: Confirmation of Improved Cognitive Function in 5×FAD MouseIntranasally Treated with Antagomir (AM)-485-3p

-   -   (1) Research Methods

Y-maze and passive avoidance tests were carried out to investigatewhether the intranasal treatment of AM-485-3p(2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)) improvedthe cognitive function of 5×FAD.

1) Y-Maze Test

A Y-maze test apparatus is composed of Y-shaped maze prepared with blackacrylic plates (10 cm wide, 41 cm long, 25 cm high). The maze isarranged with an angle of 120°. After dividing each maze into A, B and Czones, the experimental animals were placed carefully in each zone andallowed to move freely for 8 minutes. Spontaneous alternation (%) wasevaluated by measuring the number and sequence of entries into eachmaze. The entrance into the three different zones in sequence was givenone point (actual alternation, e.g., A-B—C, B—C-A, C-A-B, etc.). Nopoint was given to discontinuous entrance. The spontaneous alternation(%) was calculated by the following formula.

% Spontaneous alteration=total number of alternation/(total number ofentries−2)×100

2) Passive Avoidance Test

The passive avoidance test is a widely used method for measuring theworking memory ability of rodents. A passive avoidance test apparatus isa shuttle box divided into two chambers, one equipped with a light bulbto create a bright environment that the test animals dislike, and theother with light blocked to create an environment which is comfort forthe animals. After two hours of stress application, the passiveavoidance response was tested (training test). Aluminum grids wereplaced on the floor of the dark chamber at regular intervals so as toapply electric shocks to the sole of the animals. The experimentalanimals tend to enter the dark chamber. After keeping the animal in thebright chamber and then allowing to enter the dark chamber, electricshock (5 V, 0.5 mA, 10 sec) was applied so that it could remember it. 24hours later, the time (latency time) lapsed until the entry into thedark chamber was measured up to 90 seconds without applying electricshock (retention tests 1, 2 and 3).

(2) Research Results

FIGS. 11A-11B show the results of comparing the cognitive function ofthe 5×FAD intranasally treated with AM-485-3p(2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)). As aresult, both the spontaneous alteration and the latency time weredecreased in the 5×FAD and the control mouse as compared to WT. Becausethe typical symptoms of Alzheimer's dementia are behavior disorder andmemory decline, the behavior disorder of 5×FAD seems to be due to theexcessive accumulation and pathology of Aβ. However, the groupintranasally treated with AM-485-3p showed significant increase in boththe spontaneous alteration (FIG. 11 A) and the latency time (FIG. 11 B)as compared to 5×FAD. It means that the treatment with AM-485-3p canimprove the main symptoms of Alzheimer's by relieving the pathologicalsymptoms such as behavioral disorder and memory decline caused by theproduction of Aβ42 facilitated by miR-485-3p. Therefore, the preparationof a drug that regulates miR-485-3p or a composition thereof can be anew strategy to improve the main symptoms of Alzheimer's dementia, i.e.,behavioral disorder and cognitive function.

Example 10: Statistical Analysis

Two groups were compared by the Student's t-test, and three or moregroups were compared by the Krushall-Wallis test. When the P valueobtained from the Krushall-Wallis test was <0.05, two groups were testedpost-hoc by the Mann-Whitney U test. P value of 0.05 or smaller for thetwo-tailed test was considered statistically significant.

INDUSTRIAL APPLICABILITY

According to the present disclosure, a composition for treating a braindisease, which contains a miR-485-3p inhibitor, can restore the ELAVL2protein unlike the exiting therapeutic agents for Alzheimer's disease,which are limited only to alleviating symptoms by inducing decreasedexpression of amyloid beta 42. Therefore, it can fundamentally treatvarious diseases caused by decreased expression of ELAVL2, such asAlzheimer's disease, autism spectrum disorder, mental retardation,amyotrophic lateral sclerosis, etc. Accordingly, the present disclosureis useful for treating brain diseases including Alzheimer's diseasefundamentally.

While the specific embodiments of the present disclosure have beendescribed in detail above, those skilled of ordinary skill in the artwill appreciate that the specific embodiments are merely specificexemplary embodiments and the scope of the present disclosure is notlimited by them. It is to be understood that the substantial scope ofthe disclosure is defined by the appended claims and their equivalents.

1. A method of treating Alzheimer's disease in a subject in need thereofcomprising administering a miR-485-3p inhibitor to the subject, whereinthe miR-485-3p inhibitor is a nucleic acid molecule and inhibitsexpression of miR-485-3p.
 2. (canceled)
 3. The method of claim 1,wherein the miR-485-3p inhibitor binds to a base sequence of SEQ ID NO 1or SEQ ID NO
 2. 4. The method of claim 1, wherein the nucleic acidmolecule is a DNA, an RNA, an antagomir, a siRNA, a shRNA or anoligonucleotide.
 5. The method of claim 4, wherein the oligonucleotideis an antisense oligonucleotide comprising a sequence complementary tothe base sequence of SEQ ID NO:
 1. 6. The method of claim 5, wherein theantisense oligonucleotide comprises a base sequence selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, and SEQ ID NO:
 7. 7. The method of claim 5, wherein the antisenseoligonucleotide comprises at least one LNA (locked nucleic acid), atleast one PNA (peptide nucleic acid), at least one substitution of an—OH group at a 2′ carbon of a nucleotide with a —CH₃ (methyl), at leastone phosphorothioate bond, or combinations thereof.
 8. The method ofclaim 1, wherein the administering of the miR-485-3p inhibitor increasesan expression level of ELAVL2, inhibits production of amyloid beta 42(Aβ42), inhibits expression of amyloid precursor protein (APP), inhibitsphosphorylation of a tau protein, or combinations thereof.
 9. (canceled)10. The method of claim 1, wherein the miR-485-3p inhibitor isformulated in a composition comprising a pharmaceutically acceptablecarrier. 11-13. (canceled)
 14. The method of claim 1, wherein themiR-485-3p inhibitor is administered via intranasal administration,intravenous administration, subcutaneous injection, intrathecalinjection, inhalation administration, or oral administration.
 15. Themethod of claim 6, wherein the antisense oligonucleotide comprises atleast one base with a 2′-methoxy group, at least one base with a3′-cholesterol, at least one phosphorothioate bond, or combinationsthereof.
 16. A method of treating Alzheimer's disease in a subject inneed thereof comprising administering a miR-485-3p inhibitor to thesubject, wherein the miR-485-3p inhibitor comprises an antisenseoligonucleotide that binds to the sequence as set forth in SEQ ID NO: 1or SEQ ID NO:
 2. 17. The method of claim 16, wherein the antisenseoligonucleotide comprises the nucleotide sequence as set forth in SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:
 7. 18.The method of claim 16, wherein the antisense oligonucleotide is 10 to40 nucleotides.
 19. The method of claim 16, wherein the antisenseoligonucleotide comprises the sequence as set forth in SEQ ID NO:
 9. 20.The method of claim 19, wherein the antisense oligonucleotide is2′-O-methylated.
 21. A method of treating Alzheimer's disease in asubject in need thereof comprising administering a miR-485-3p inhibitorto the subject, wherein the miR-485-3p inhibitor comprises an antisenseoligonucleotide that comprises the nucleotide sequence as set forth inSEQ ID NO:
 7. 22. The method of claim 21, wherein the antisenseoligonucleotide is 2′-O-methylated.
 23. The method of claim 1, whereinthe miR-485-3p inhibitor comprises the nucleotide sequence as set forthin SEQ ID NO:
 7. 24. The method of claim 16, wherein the miR-485-3pinhibitor comprises the nucleotide sequence as set forth in SEQ ID NO:7.